Method of judging homogenization/reaction completion and method of measuring solution concentration using the same

ABSTRACT

It is intended to provide a method for determining homogenization and/or reaction completion, capable of making the measurement time necessary and sufficient and enhancing the measurement speed, and a method for measuring solution concentration using the same. According to the method for determining homogenization and/or reaction completion and the method for measuring solution concentration using the same, homogenization and reaction completion are determined based on the optical property of the liquid mixture of a test liquid and a reagent liquid.

TECHNICAL FIELD

The present invention relates to solution concentration measuringmethods and solution concentration measuring apparatuses for determiningthe concentration of a solute dissolved in a test liquid, for example,protein. More specifically, the present invention measures theconcentration of a specific component contained in a test liquid, bymixing a reagent liquid into the test liquid to change the opticalproperty of the test liquid deriving from only the specific component.Particularly, by mixing the test liquid and the reagent liquid tocoagulate the protein component, and detecting the decrease in lighttransmitted through the test liquid after the mixing and/or the increasein the intensity of light scattered during the propagation through thetest liquid, the present invention measures the concentration of thisprotein component.

The present invention determines that the test liquid and the reagentliquid have been sufficiently stirred until homogenized, when therelation between the elapsed period of time since the mixing and theintensity of light transmitted through the liquid mixture or theintensity of scattered light satisfies a predetermined condition. Also,by this, the present invention can simultaneously determine that thereaction between the test liquid and the reagent liquid has beencompleted. In this way, by determining homogenization and/or reactioncompletion, it is possible to set the measurement time necessary andsufficient, and to reduce the measurement time. In the case of notcontrolling the temperature of the liquid mixture of the test liquid andthe reagent liquid, in particular, the present invention can realizehigh reliability with a necessary and sufficient measurement time andprovide highly practical methods for measuring solution concentration.

BACKGROUND ART

According to conventional methods of measuring solution concentration, atest liquid and a reagent liquid are mixed in a predetermined volumeratio and sufficiently stirred until the resultant mixture becomeshomogeneous, to prepare a liquid mixture. Then, the liquid mixture isstirred at a predetermined temperature, and upon the lapse of apredetermined period of time, the optical property of the liquid mixtureis measured to determine the concentration. In methods of measuring theconcentration of a specific component by utilizing biochemicalreactions, such as enzyme reactions and antigen-antibody reactions, itis common to set the predetermined temperature to 37° C., which is closeto living body temperature. It is also common to set the predeterminedperiod of time to a period of time within which the reactionsufficiently reaches completion. Naturally, since the rate of a reactiondepends on temperature, concentration, and the like, a sufficient periodof time for the completion of the reaction is set in consideration ofthe concentration of the test liquid at the predetermined temperature.

As described above, in conventional practice, optical property ismeasured under conditions where the liquid mixture is sufficientlystirred until homogenized and the reaction would never fail to reachcompletion. That is, sufficient conditions for homogenization andreaction completion are set.

Also, with conventional apparatuses for measuring solutionconcentration, a test liquid is retained in a sample cell that isstructured to propagate light through the test liquid. This sample cellis in the form of a rectangular parallelepiped, made of, for example,glass, and has transmission faces that are transparent. Thus, light canbe propagated through the test liquid. When the test liquid and areagent liquid are introduced into the sample cell and mixed, the samplecell is detached from the optical system for measuring optical property,and the following operations are performed.

Usually, the top part of this sample cell is open, and a predeterminedvolume of a test liquid is introduced from the top part using a dropper,a pipette, a syringe, or the like. Subsequently, a predetermined volumeof a reagent liquid is mixed thereinto such that the volume ratio of thetest liquid to the reagent liquid is constant. Thereafter, the resultantmixture is sufficiently stirred in the sample cell with a stirringstick, stirrer, or the like until it becomes homogenized, and the wholesample cell is kept at a predetermined temperature, for example, in aconstant-temperature bath. After the lapse of a predetermined period oftime, the sample cell is remounted onto the optical system, and theoptical property of the liquid mixture in the sample cell is measured.

However, there have been problems in that conventional solutionconcentration measuring methods involve a large number of processes andconventional solution concentration measuring apparatuses arelarge-scaled. Further, there has been another problem of requiringincreased measurement time. Therefore, there is a demand for solutionconcentration measuring apparatuses having a simple structure without aconstant-temperature bath and the like, as well as solutionconcentration measuring methods capable of easy automation.

Further, there has also been another problem in that the processes ofloading and unloading the sample cell cause a slight change in theposition of the optical system, possibly leading to errors inmeasurement results. Furthermore, still another problem has been thatcomplicated operations are necessary, and hence, operation mistakes,etc. tend to occur, thereby resulting in poor reliability.

In consideration of the above-mentioned problems, an object of thepresent invention is to provide a highly reliable method of measuringsolution concentration capable of easy automation, as well as a highlyreliable, small-sized apparatus of measuring solution concentrationcapable of easy automation. The present invention further provides asolution concentration measuring method and a solution concentrationmeasuring apparatus which can reduce the time necessary forhomogenization and/or reaction completion to the requisite minimum,thereby enabling a reduction in measurement time.

DISCLOSURE OF INVENTION

The present invention relates to a method for determining homogenizationand/or reaction completion, including the steps of: (1) mixing a testliquid and a reagent liquid to obtain a liquid mixture; (2) measuring anoptical property of the liquid mixture after the mixing continuously ora plurality of times discretely; (3) obtaining a relation between themeasured value of the optical property obtained and the elapsed periodof time since the start of the measurement after the mixing; and (4)determining, on the basis of the relation, whether the test liquid andthe reagent liquid have been substantially homogeneously mixed with eachother and/or a reaction between the test liquid and the reagent liquidhas been substantially completed. The steps (1) to (4) are performed inthis order.

In this method for determining homogenization and/or reactioncompletion, the step (3) is preferably a step of obtaining dS1/dt(wherein S1 is the measured value of the optical property obtained and Tis the elapsed period of time since the start of the measurement afterthe mixing), and the step (4) is preferably a step of determining thatthe test liquid and the reagent liquid have been substantiallyhomogeneously mixed with each other and/or the reaction between the testliquid and the reagent liquid has been substantially completed, when thedS1/dt has continuously been in a predetermined range R1 for apredetermined period of time T1 or longer.

Also, the step (3) is preferably a step of obtaining (dS1/dt)/S1(wherein S1 is the measured value of the optical property obtained and Tis the elapsed period of time since the start of the measurement afterthe mixing), and the step (4) is preferably a step of determining thatthe test liquid and the reagent liquid have been substantiallyhomogeneously mixed with each other and/or the reaction between the testliquid and the reagent liquid has been substantially completed, when the(dS1/dt)/S1 has continuously been in a predetermined range R2 for apredetermined period of time T2 or longer.

Further, the present invention pertains to a method for determininghomogenization and/or reaction completion, including the steps of: (1)mixing a test liquid and a reagent liquid to obtain a liquid mixture;(2) measuring an optical property of the test liquid and the liquidmixture continuously, or, measuring an optical property of the testliquid at least once and measuring an optical property of the liquidmixture after the mixing a plurality of times discretely; (3) obtaininga relation between the measured value of the optical property obtainedand the elapsed period of time since the start of the measurement afterthe mixing; and (4) determining, on the basis of the relation, that thetest liquid and the reagent liquid have been substantially homogeneouslymixed with each other and/or the reaction between the test liquid andthe reagent liquid has been substantially completed. The steps (1) to(4) are performed in this order.

In the method for determining homogenization and/or reaction completion,the step (3) is preferably a step of obtaining (dS1/dt)/(S1−S0) (whereinS0 is the measured value of the optical property of the test liquid, S1is the measured value of the optical property of the liquid mixture, andT is the elapsed period of time since the start of the measurement afterthe mixing), and the step (4) is preferably a step of determining thatthe test liquid and the reagent liquid have been substantiallyhomogeneously mixed with each other and/or the reaction between the testliquid and the reagent liquid has been substantially completed, when the(dS1/dt)/(S1−S0) has continuously been in a predetermined range R3 for apredetermined period of time T3 or longer.

Furthermore, the present invention is directed to a method for measuringsolution concentration, wherein the homogenization of the mixture of thetest liquid and the reagent liquid and/or the substantial completion ofthe reaction therebetween are determined according to theabove-mentioned method for determining homogenization and/or reactioncompletion, and then the concentration of a specific component of thetest liquid is determined based on the measured value S1 or the measuredvalues of S0 and S1.

This method for measuring solution concentration preferably includes thestep of mixing another reagent liquid with the test liquid, afterdetermining that the test liquid and the reagent liquid have beenhomogeneously mixed and/or the reaction therebetween has beensubstantially completed.

In this case, preferably, another reagent liquid is mixed with the testliquid upon the lapse of a predetermined period of time T4 afterdetermining that the test liquid and the reagent liquid have beenhomogeneously mixed and/or the reaction therebetween has beensubstantially completed, and the optical property of the liquid mixtureis measured prior to the lapse of the predetermined period of time T4.

The present invention also relates to an apparatus for measuringsolution concentration, including: a light source that irradiates a testliquid with light; a sample cell that retains the test liquid; aphotosensor 1 that detects light transmitted through the test liquidand/or a photosensor 2 that detects light scattered while the light ispropagated through the test liquid; and a computer that analyzes anoutput signal of the photosensor 1 and/or the photosensor 2, whereinbased on the above-mentioned method for measuring solutionconcentration, the computer analyzes the output signal of thepohotosensor 1 and/or the photosensor 2 to calculate the concentrationof the test liquid.

That is, the computer preferably includes controlling means formeasuring an optical property of the liquid mixture obtained by mixing atest liquid and a reagent liquid continuously or a plurality of timesdiscretely, obtaining a relation between the measured value of theoptical property obtained and the elapsed period of time since the startof the measurement after the mixing, determining, on the basis of therelation, that the test liquid and the reagent liquid have beensubstantially homogeneously mixed with each other and/or a reactionbetween the test liquid and the reagent liquid has been substantiallycompleted, and determining the concentration of a specific component inthe test liquid based on the measured value.

Also, the controlling means of the computer may measure the opticalproperty of the test liquid and the liquid mixture continuously, or, itmay measure the optical property of the test liquid at least once andmeasure the optical property of the liquid mixture after the mixing aplurality of times discretely.

Further, it is preferred that the apparatus for measuring solutionconcentration include an injector that injects a reagent liquid into thetest liquid in the sample cell for mixing and that the injector becontrolled by the computer or the controlling means.

In the apparatus for measuring solution concentration, it is preferredthat the optical property of the test liquid be measured, using thelight source, to determine the concentration of a specific component inthe test liquid.

It is also preferred to perform stirring by means of the mechanicaleffect produced by the injection of the reagent liquid.

Further, in the above-mentioned method for determining homogenizationand/or reaction completion, method for measuring solution concentration,and apparatus for measuring solution concentration, it is preferred thata measurement be rendered invalid when homogenization and/or reactioncompletion has not been determined within a predetermined period of timeT from the start of the measurement.

It is also preferred that when the concentration of the analyte in thetest liquid is the lowest possible concentration, the above-mentionedpredetermined period of time T satisfy the relation T≧T5 wherein T5 isthe elapsed period of time since the start of a measurement untilhomogenization or reaction completion is determined by theabove-mentioned method for determining homogenization and/or reactioncompletion.

It is also preferred that the substance that reacts with the analyte bean antibody that specifically reacts and combines with the analyte andthat the signal related to the optical property that derives from thespecific binding reaction be the turbidity of the liquid mixture.

It is further preferred that the analyte be human albumin.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a top view of a solution concentration measuring apparatusaccording to Embodiment 1 of the present invention;

FIG. 2 is a partially cross-sectional side view of the solutionconcentration measuring apparatus according to Embodiment 1 of thepresent invention;

FIG. 3 is a graph showing the changes over time in the output signal ofa photosensor 5 in Embodiment 3 of the present invention;

FIG. 4 is a graph showing the rate of change over time in the derivativesignal of output signal of a photosensor 5 in Embodiment 1 of thepresent invention;

FIG. 5 is a graph obtained by enlarging the ordinate around 0 in FIG. 4;

FIG. 6 is a graph obtained by enlarging the ordinate at 0 and greaterand enlarging the abscissa around 14.5 to 16.5 seconds in FIG. 4;

FIG. 7 is a graph obtained by enlarging the ordinate around 0 andenlarging the abscissa around 17 to 20 seconds in FIG. 4;

FIG. 8 is a top view of a solution concentration measuring apparatusaccording to Embodiment 2 of the present invention;

FIG. 9 is a partially cross-sectional side view of the solutionconcentration measuring apparatus according to Embodiment 2 of thepresent invention;

FIG. 10 is a graph showing the changes over time in the output signal ofa photosensor 12 in Embodiment 2 of the present invention;

FIG. 11 is a graph showing the rate of change over time in thederivative signal of output signal of the photosensor 12 in Embodiment 2of the present invention;

FIG. 12 is a graph obtained by enlarging the ordinate around 0 andenlarging the abscissa around 60 to 200 seconds in FIG. 11;

FIG. 13 is a graph showing the values obtained by dividing thederivative signal of output signal of a photosensor 12 of Embodiment 3of the present invention by the output signal;

FIG. 14 is a graph obtained by enlarging the ordinate around 0 andenlarging the abscissa around 60 to 200 seconds in FIG. 13;

FIG. 15 is a graph in which the ordinate of FIG. 12 is logarithmicallyexpressed;

FIG. 16 is a graph showing the dependency of the output signal of thephotosensor 12 used in Embodiments 2 to 3 of the present invention onthe protein concentration;

FIG. 17 is a top view of a solution concentration measuring apparatusaccording to Embodiment 5 of the present invention; and

FIG. 18 is a graph showing the changes over time in the output signal ofa photosensor 12 in Embodiment 5 of the present invention.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention relates to a solution concentration measuringmethod that qualitatively or quantitatively determines an analyte, bymixing a test liquid containing the analyte with a reagent liquidcontaining a substance that reacts with the analyte, and detecting asignal related to the optical property deriving from the reaction.

The present invention relates to a method for determining homogenizationand/or reaction completion, including the steps of: (1) mixing a testliquid and a reagent liquid to obtain a liquid mixture; (2) measuring anoptical property of the liquid mixture after the mixing continuously ora plurality of times discretely, or, measuring an optical property ofthe test liquid and the liquid mixture continuously, or, measuring anoptical property of the test liquid at least once and measuring anoptical property of the liquid mixture after the mixing a plurality oftimes discretely; (3) obtaining a relation between the measured value ofthe optical property obtained and the elapsed period of time since thestart of the measurement after the mixing; and (4) determining, on thebasis of the relation, whether the test liquid and the reagent liquidhave been substantially homogeneously mixed with each other and/or areaction between the test liquid and the reagent liquid has beensubstantially completed.

The present invention also provide a solution concentration measuringmethod and a solution concentration measuring apparatus using thismethod for measuring homogenization and/or reaction completion.

Referring now to drawings, various embodiments of the present inventionare described below.

Embodiment 1

Referring to FIGS. 1 and 2, Embodiment 1 of the present invention isdetailed below. FIG. 1 is a top view of a solution concentrationmeasuring apparatus according to Embodiment 1 of the present invention.FIG. 2 is a partially cross-sectional side view of the solutionconcentration measuring apparatus according to Embodiment 1 of thepresent invention. In FIGS. 1 and 2, the skeletal part of a sample cell1 is composed of an aluminum container that is in the form of arectangular parallelepiped and has an opening at the top. A glass plate,serving as an optical window, is fitted into a pair of side faces of thesample cell 1 to form an optical path, so that light can be transmittedthrough a test liquid (or a liquid mixture of a test liquid and areagent liquid) retained in the sample cell 1. In FIG. 1, the distancebetween the optical windows (optical length), which is the distance inthe direction of light propagation in the sample cell 1, is representedby A, and the distance in the direction perpendicular to the directionof light propagation in the sample cell 1 is represented by B. In thisembodiment, A and B are set to 0.8 cm and 0.4 cm, respectively, as arepresentative example to explain the present invention.

As illustrated in FIG. 1, an injection port 2 is provided in the edge ofthe side face of the sample cell 1 having no optical window, and theinternal diameter (diameter) of the injection port 2 is 0.1 cm. Asillustrated in FIG. 2, the center of the vertical section of theinjection port 2 is positioned at a distance x from the bottom face ofthe sample cell 1 and at a distance z from the optical window. Injectiondirection 10 is parallel to the faces having the optical windows andperpendicular to the direction of light propagation. In this embodiment,x and y are set to 0.4 cm and 0.1 cm, respectively, as a representativeexample to explain the present invention.

A semiconductor laser module 3, which is a light source, projectssubstantially parallel light 4 onto the test liquid in the sample cell.The substantially parallel light 4 has a wavelength of 780 nm, anintensity of 3.0 mW, and a beam diameter of 0.2 cm. The optical axis ofthis substantially parallel light 4 is parallel to the bottom face ofthe sample cell 1 and positioned at a distance of 0.4 cm from the bottomface. Therefore, the optical axis and the injection port 2 arepositioned at the same height from the bottom face, and the optical axisof the substantially parallel light 4 and the injection axis extendingfrom the center of the section of the injection port 2 in the injectiondirection 10 have a point of intersection in the solution in the samplecell 1.

A photosensor 5 is a photosensor which detects light that has beentransmitted through the test liquid. A pump 6 injects a reagent liquidfrom the injection port 2 into the test liquid in the sample cell 1. Acomputer 7 analyzes the output signal of the photosensor 5 and controlsthe pump 6. An arrow 8 schematically indicates the direction of thevortex that occurs in the sample cell 1 when the reagent liquid isinjected from the injection port 2. Also, the lowest part of liquidlevel 9 of the test liquid is positioned at a height h from the bottomface of the sample cell 1. In the present invention, the liquid level isdefined as the level that is in contact with the lowest part of theliquid level 9 and parallel to the horizontal level. According to thisdefinition, the injection direction is parallel to the liquid level inthis embodiment.

The sample cell 1 has inner wall corners that are rounded. That is, thecorners of the sample cell 1 are not right-angled in a strict sense.Thus, when h=0.8 cm, the sample cell 1 contains about 0.25 ml of thetest liquid.

In this embodiment, a dispersion obtained by homogeneously dispersingpolystyrene fine particles having a mean diameter of 20 nm in pure wateris charged into the sample cell 1 as the test liquid. The whole testliquid is homogeneously turbid.

First, the mechanism of injecting pure water into this test liquid isdescribed. The polystyrene fine particles have a specific gravity closeto that of pure water, and their particle size is also small. Thus, oncethey have been fully homogeneously dispersed in pure water, phenomenasuch as separation and precipitation do not occur during the period oftime in which the method according to the present invention is carriedout. However, if they have not been homogeneously dispersed due toinsufficient stirring, such phenomena may occur.

When pure water is injected into this test liquid, the polystyrene fineparticles diffuse therethrough, so that the concentration of thepolystyrene fine particles lowers. Consequently, the degree of opacityof the whole test liquid, i.e., turbidity, lowers. This turbidity ismeasured as the intensity of transmitted light by detecting the outputsignal of the photosensor 5.

In this way, the change in the turbidity of the liquid containing fineparticles due to diffusion involves no chemical reaction. Therefore, theturbidity of the whole test liquid depends solely on the degree ofdiffusion of the polystyrene fine particles, and hence, there is no needto take reaction speed into consideration. That is, the fact that theturbidity has been stabilized at a certain value means that the fineparticles have been sufficiently spread throughout the liquid andhomogeneously dispersed.

From these, observing the turbidity by mixing a liquid containing fineparticles as a reagent liquid with a test liquid is convenient inverifying the stirring effect. In this embodiment, the present inventionis simplified for the purpose of explanation by selecting only theresult of determination of the homogenization attained by the stirringof the present invention.

The operations of this embodiment were conducted as follows:

First, the test liquid containing the polystyrene fine particles wasintroduced into the sample cell 1. At this time, the volume of the testliquid introduced was 0.25 ml. The computer 7 was started to measure(record) the output signal of the photosensor 5. The changes over timein the output signal of the photosensor 5 after the start of themeasurement following the introduction are shown in FIG. 3.

In FIG. 3, the elapsed period of time since the start of measurement ofthe output signal is plotted in abscissa, and the output signal of thephotosensor 5 is plotted in ordinate. At an elapsed time of 10 seconds,the computer 7 controlled the pump 6 so that pure water was injectedfrom the injection port 2 over 2 seconds. The changes in the outputsignal of the photosensor 5 in the case of pure water injection, asdescribed, are shown by solid lines in FIG. 3. In FIG. 3, “a”, “b”, “c”,and “d” show the changes in the output signal of the photosensor 5resulting from the injection of 0.1 ml of pure water, 0.07 ml of purewater, 0.05 ml of water, and 0.03 ml of pure water, respectively.

In this figure, for 2 to 3 seconds from the start of the pure waterinjection, i.e., the elapsed time of 10 seconds since the start of therecording, the flux of the injected pure water itself entered theoptical path of the substantially parallel light 4. As a result, theintensity and the propagation direction of transmitted light weredisturbed, causing a violent change in the output signal of thephotosensor 5. In FIG. 3, the amplitude of the change is illustrated asthe hatched area, and the output signal changed between 0.6 to 1.4 V.Even when the same volume of pure water was injected, the amplitude ofthe change was shown in this area, though the details of the change werenot confirmed. Since the concentration of the polystyrene particleslowered depending on the amount of pure water injected, the turbidityalso decreased depending on the amount of injection.

When the injected amounts of pure water were 0.1 ml, 0.07 ml, and 0.05ml, as shown by the solid lines “a”, “b”, and “c” of FIG. 3,respectively, the resultant output signals were commensurate with therespective amounts of injection, and the output signals themselves werestabilized. This is because the injection of pure water produced avortex in the test liquid, which allowed the liquid mixture of the testliquid and the pure water to be sufficiently stirred until it becamehomogenized. The homogenization could also be confirmed by visualobservation. On the other hand, when the amount of injection was 0.03ml, the output signal was not stabilized, as shown by the solid line“d”. This is because the liquid mixture of the test liquid and the purewater was stirred insufficiently and was not stirred until homogenized.The inconsistencies in the concentration of the polystyrene fineparticles could also be confirmed by visual observation.

As described above, the solid lines “a” to “c” represented the caseswhere the liquid mixture of the test liquid and the pure water wassufficiently stirred until homogenized, while the solid line “d”represented the case where the liquid mixture could not be sufficientlystirred until homogenized.

Conventionally, in measuring the optical property of a test liquid, suchas turbidity, from the output signal of the photosensor 5, one had towait for the lapse of a period of time within which the output signal ofthe photosensor 5 became sufficiently stabilized. After the lapse ofthat period of time, one analyzed the output signal of the photosensor5. For example, the output signal of the photosensor 5 at an elapsedtime of 60 seconds since the start of the recording was used. In thecase of the solid line “d” according to the present invention, however,an accurate measurement of the optical property was not possible,because the liquid mixture had not been homogenized. Hence, there was aneed to additionally make visual observations to confirm thehomogenization.

Contrary to such conventional art, the method of the present invention,which will be described below, determines whether or not the liquidmixture has been sufficiently stirred, i.e., whether or not the liquidmixture has become homogenized, based on the optical property of theliquid mixture, i.e., the output signal of the photosensor 5. Summingup, this method is an example of being able to reduce the measurementtime by setting the waiting time from the start of a measurement untilobtaining the result necessary and sufficient.

First, the maximum period of time within which a measurement isperformed is set in advance. This period refers to the longest waitingperiod of time from the start of the measurement until obtaining theresult, and if a measurement takes more than this maximum period oftime, this measurement is rendered invalid. This pre-set period of timeis referred to as a predetermined period of time T.

According to this method, homogenization is determined when the amountof change in an output signal S1 of the photosensor 5 per unit time,i.e., a derivative signal dS1/dt, has continuously been in apredetermined range R1 for a predetermined period of time T1 within thepredetermined period of time T.

Specifically, the liquid mixture is determined as having beenhomogenized when dS1/dt [V/S] has continuously been in a predeterminedrange R1 represented by formula (1) for a predetermined period of timeT1 (1.5 seconds) or longer within a predetermined period of time T (60seconds) after the start of a measurement.−5×10⁻⁴ ≦dS1/dt≦5×10⁻⁴  (1)It should be noted that if the predetermined period of time T, whichcorresponds to the longest waiting period of time, is not properly set,the derivative signal dS1/dt may be in the predetermined range R1 forthe predetermined period of time T1, even if the liquid mixture has notbeen homogenized due to insufficient stirring, particularly even if purewater regions are separated from fine particle regions. Thus, there is apossibility of making a wrong determination that the liquid mixture hasbeen homogenized.

FIG. 4 shows the derivative signal of output signal of the photosensor5. The solid lines “a” to “d” of FIG. 3 correspond to the derivativevalues (dS/dt) of the signal intensities represented by the solid lines“a” to “c” and the dotted line “d” of FIG. 4, respectively. In FIG. 4,in the same manner as in FIG. 3, during the period of about 2 seconds ormore from the elapsed time of 10 seconds at which the injection of purewater was started, the flux of the injected pure water itself enteredthe optical path of the substantially parallel light 4. As a result, theintensity and the propagation direction of transmitted light weredisturbed, causing a violent change in the derivative signal of outputsignal of the photosensor 5. In FIG. 4, the solid lines “a” to “c”appeared to coincide with one another. Thus, a graph was prepared byenlarging the ordinate of FIG. 4 around 0 (FIG. 5). FIG. 5 showed thatthe solid lines “a” to “c” asymptotically approached zero, and that thebroken line “d” largely swayed toward the minus side before goingthrough the minimum value. However, in FIG. 5, the solid lines “a” to“c” also appeared to coincide with one another.

Thus, FIG. 6 showed a graph obtained by enlarging the ordinate at zeroand greater and enlarging the abscissa around 14.5 to 16.5 seconds inFIG. 4. In FIG. 6, ▴ corresponds to “a”, ▪ corresponds to “b”, and ●corresponds to “c”. As is clear from FIG. 6, “a” to “c” asymptoticallyapproached zero.

For the condition for determining homogenization, a point of time whenthe derivative signal dS1/dt of output signal of the photosensor 5 hascontinuously been in the predetermined range R1 represented by formula(1) for the predetermined period of time T1 (1.5 seconds) or longerwithin the predetermined period of time T (60 seconds) after the startof a measurement was found as follows.

The derivative signal dS1/dt of output signal of the photosensor 5became 5×10⁻⁴ [V/S] or less from an elapsed time of 14.79 seconds for“a”, from an elapsed time of 14.88 seconds for “b”, and from an elapsedtime of 14.92 seconds for “c”. After these points in time, since “a” to“c” asymptotically approached zero, the derivative signal was in thepredetermined range R1 as expressed by formula (1).

Therefore, in FIG. 6, if the point of the lapse of 1.5 seconds from thepoint when the derivative signal dS/dt fell within the predeterminedrange R1 expressed by formula (1) is within the predetermined period oftime (T=60 seconds) from the start of the measurement, the liquidmixture could be determined as having been homogenized at an elapsedtime of 15 seconds. Specifically, for “a”, homogenization could bedetermined at an elapsed time of 16.29 seconds. For “b”, homogenizationcould be determined at an elapsed time of 16.38 seconds. For “c”,homogenization could be determined at an elapsed time of 16.42 seconds.

Meanwhile, a graph was prepared by enlarging the ordinate of FIG. 4around 0 and enlarging the abscissa at elapsed times of 17 to 20 seconds(FIG. 7). As shown in FIG. 7, the derivative signal dS1/dt of outputsignal of the photosensor 5 is in the predetermined range R1 expressedby formula (1) from an elapsed time of 17.69 seconds until an elapsedtime of 18.71 seconds. In this case, since the derivative signal waswithin the R1 only for 1.02 seconds (=18.71−17.69), the liquid mixturecould not be determined as having been homogenized. Also, the liquidmixture according to “d” was determined as not having been homogenized,because, as is clear from FIG. 5, the derivative signal does not fallwithin the range R1 at least until the point of the lapse of thepredetermined period of time (T=60 seconds) after the start of themeasurement. By using such determination condition, it was possible toadequately determine whether or not the liquid mixture had beensufficiently stirred and homogenized.

In measuring the optical property of a test liquid, such as turbidity,from the output signal of the photosensor 5, the output signal of thephotosensor 5 at the point when homogenization was determined may beanalyzed, as described above. Specifically, for “a”, the output signalof the photosensor 5 at the elapsed time of 16.29 seconds may be used,and for “b”, the output signal of the photosensor 5 at the elapsed timeof 16.38 seconds may be used. Also, for “c”, the output signal of thephotosensor 5 at the elapsed time of 16.42 seconds may be used.Accordingly, the optical property can be measured in a necessary andsufficient measurement time while the accuracy is assured, so that themeasurement time can be reduced. Further, misoperations due toinsufficient homogenization can be avoided.

It is needless to say that the condition for determining homogenizationis not limited to the above-described condition. That is, T, T1, and thepredetermined range R expressed by formula (1) may be set asappropriate, according to various conditions such as the size of thefine particles, the density of the particle, the injection speed, thearrangement of the optical system, the accuracy required, themeasurement time, the calibration curb, etc. Also, in calculating theconcentration of a specific component of a test liquid, the computer 7analyzes the output signal of the photosensor 5 at the point whenhomogenization was determined, while referring to the pre-setcalibration curb, in order to calculate the concentration of the testliquid.

As described above, according to this embodiment, the degree of stirringof the liquid mixture and homogenization can be determined with thesample cell mounted on the optical system. Further, since the timeneeded for a measurement is necessary and sufficient, time can be saved.Accordingly, while the process can be simplified, misoperations areunlikely to occur. These practical effects are extremely large, therebymaking it possible to enhance the efficiency of measurements and testsand to achieve labor-savings thereof.

Embodiment 2

Referring now to FIGS. 8 and 9, Embodiment 2 of the present invention isdescribed in detail below. In FIGS. 8 and 9, the constituent elementsrepresented by reference characters 1 to 10 are the same as theconstituent elements represented by reference characters 1 to 10 inFIGS. 1 and 2 used for describing the foregoing Embodiment 1, and theyfunction in the same manner. It should be noted, however, that the lightprojected and scattered in a test liquid or a liquid mixture is detectedin this embodiment.

Scattered light 11 that arises while the substantially parallel light 4is propagated through a test liquid is detected by a photosensor 12. Theoutput signal of the photosensor 12 corresponds to the intensity of thescattered light 11 and is analyzed by the computer 7.

In this embodiment, the concentration of protein in a test liquid ismeasured, using a solution containing protein as the test liquid and areagent liquid of sulfosalicylic acid (a reagent obtained by dissolvingsodium sulfate in an aqueous solution of 2-hydroxy-5-sulfobenzoic acid)as the reagent liquid. In this case, when the test liquid and thereagent liquid of sulfosalicylic acid are mixed together, the proteincomponent of the test liquid coagulates, thereby making the resultantwhole liquid mixture turbid. Thus, by measuring the degree of opacity,i.e., turbidity, the protein concentration can be determined. Theturbidity is measured as the intensity of scattered light, i.e., theoutput signal of the photosensor 12. The higher the proteinconcentration, the higher the turbidity, and the greater the outputsignal of the photosensor 12.

In calculating the protein concentration, the turbidity of a standardsolution of a known concentration, i.e., the output signal of thephotosensor 12, is measured in advance, and based on this, a calibrationcurb is prepared. Then, the turbidity of a test liquid of an unknownconcentration is measured, and the concentration is calculated byreferring to the prepared calibration curb.

In this embodiment, the optical property, i.e., the property change inturbidity, is influenced not only by the stirring effect but also by thereaction (coagulation) property, unlike the foregoing Embodiment 1.

The operations of this embodiment were performed as follows:

First, an aqueous solution with a protein concentration of 100 mg/dl wasintroduced as a test liquid into the sample cell 1. At this time, thevolume of the test liquid introduced was 0.25 ml. The computer 7 wasstarted to record the output signal of the photosensor 12. The changesover time in the output signal of the photosensor 12 after the start ofthe recording following the introduction are shown by ● in FIG. 10.

In FIG. 10, the elapsed period of time since the start of measurement ofthe output signal is plotted in abscissa, and the output signal of thephotosensor 12 is plotted in ordinate. At an elapsed time of 20 secondssince the start of the measurement, the computer 7 controlled the pump 6so that 0.05 ml of the reagent liquid of sulfosalicylic acid wasinjected from the injection port 2 over 2 seconds.

Likewise, 0.25 ml of an aqueous solution with a protein concentration of30 mg/dl, 10 mg/dl, or 0 mg/dl was introduced into the sample cell 1. Atan elapsed time of 20 seconds since the start of the measurement, 0.05ml of the reagent liquid of sulfosalicylic acid was injected. The outputsignals of the photosensor 12 for the aqueous solutions having theconcentrations of 30 mg/dl, 10 mg/dl, and 0 mg/dl, respectively, areshown by ▪, X, and ◯ in FIG. 10.

In FIG. 10, the output signals as shown by ●,▪, X, and ◯ changed largelynear the point when the reagent liquid was injected, and this wasattributed to the invasion of the flux of the injected reagent liquiditself on the optical path of the substantially parallel light 4. Thiswas because the refractive index of the protein aqueous solution, whichis a test liquid, is different from that of the aqueous solution ofsulfosalicylic acid, and hence, the resultant local unevenness caused alarge change in the intensity of the scattered light. Further, this wasalso because the injection caused fine particles such as bubbles toenter the optical path, thereby causing a large change in the intensityof the scattered light. The region of this large change was shown byhatching. From the start of the measurement until the elapsed time of 20seconds at which the injection was started, all of the respective solidlines and the dotted line coincided with one another, so this period wasillustrated as only one solid line.

In calculating the concentration of a specific component of a testliquid, the computer 7 analyzes the output signal of the photosensor 12after the mixing of the reagent liquid represented by this solid line,while referring to the previously prepared calibration curb, in order tocalculate the concentration of the test liquid. In the above operations,since the same volume of the reagent liquid is injected into the samevolumes of the test liquids over the same period of time, thehomogenization by stirring proceeds in the same manner. However, as isclear from FIG. 10, it takes a different period of time for each outputsignal to reach saturation, i.e., to become stabilized. This is becausethe reaction speed is different according to the protein concentration.

In such cases, conventionally, the concentration was calculated, using atest liquid that required the longest period of time for thestabilization of the output signal. That is, in the case of a testliquid having a lowest protein concentration to be possibly detected,the output signal was measured at a point in time until which the outputsignal was expected to have been sufficiently stabilized, and theconcentration of the test liquid was calculated using the measuredoutput signal. Such a method has a problem of requiring a longmeasurement time even if the measurement can be completed within a shortperiod of time because of high reaction speed. Further, in the case ofhigh protein concentrations and high reaction speeds, if anunnecessarily long period of time elapses, the coagulated protein maystart precipitating to cause a change in the intensity of scatteredlight, thereby impairing the accuracy. Also, since the reaction speed isdependent on temperature as well, there was a need to perform ameasurement at a constant temperature.

Hence, in the following, this embodiment describes the method of thepresent invention that not only calculates the concentration but alsodetermines the completion of a reaction, based on the output signal ofthe photosensor 12. This embodiment can set the measurement timenecessary and sufficient, thereby enabling a substantial increase inmeasurement speed. In addition, this embodiment needs no temperaturecontrol and can prevent a degradation in accuracy due to theprecipitation phenomenon and the like. In such cases as described above,sufficient completion of a reaction (the stability of the output signal)is determined based on the optical property of the solution (the outputsignal of the photosensor 12).

First, the maximum period of time within which a measurement isperformed is set in advance. This period refers to the longest waitingperiod of time from the start of the measurement until obtaining theresult, and if a measurement takes more than this maximum period oftime, this measurement is rendered invalid. This pre-set period of timeis referred to as a predetermined period of time T.

According to this method, homogenization is determined when the amountof change in an output signal S1 of the photosensor 12 per unit time,i.e., a derivative signal dS1/dt, has continuously been in apredetermined range R1 for a predetermined period of time T1 within thepredetermined period of time T.

Specifically, homogenization is determined when dS1/dt [V/S] hascontinuously been in a predetermined range R1 represented by formula (2)for a predetermined period of time T1 (10 seconds) or longer within apredetermined period of time T (200 seconds) after the start of ameasurement.−1×10⁻⁴ ≦dS1/dt≦1×10⁻⁴  (2)

In FIG. 11, the derivative signal of output signal of the photosensor 12was plotted in ordinate. ●, ▪, and X of FIG. 11 correspond to thederivative signals represented by ●, ▪,and X of FIG. 10, respectively.In FIG. 11, in the same manner as in FIG. 10, during the period of about2 seconds or more from the elapsed time of 20 seconds at which theinjection of the reagent liquid was started, the flux of the injectedreagent liquid itself entered the optical path of the substantiallyparallel light 4. As a result, the intensity of the scattered light wasdisturbed, causing a violent change in the output signal of thephotosensor 12. In FIG. 11, ◯ was omitted, since it looks likesubstantially zero.

Since the details of FIG. 11 are not readable, a graph was prepared asFIG. 12 by enlarging the ordinate of FIG. 11 around 0 and enlarging theabscissa at 60 to 200 seconds. FIGS. 11 and 12 indicate that thederivative signal of output signal of the photosensor 12 was in theorder of ●>▪>X immediately after the injection of the reagent liquid,but after an elapsed time of about 120 seconds, this sequence wastotally reversed, so that the derivative signal was in the order ofX>▪>●. Then, all of ●, ▪, and X asymptotically approached zero.

For the condition for determining the completion of a reaction, a pointof time when the derivative signal dS1/dt of output signal of thephotosensor 12 has continuously been in the predetermined range R1represented by formula (2) for the predetermined period of time T1 (10seconds) or longer within the predetermined period of time T (200seconds) after the start of a measurement was found from FIG. 12 asfollows.

The derivative signal dS1/dt of output signal of the photosensor 12became 1×10⁻⁴ [V/S] or less from an elapsed time of 77 seconds for ●,from an elapsed time of 135 seconds for ▪, and from an elapsed time of166 seconds for X. After these points in time, since ●, ▪, and Xasymptotically approached zero, the derivative signal was in thepredetermined range R1 as expressed by formula (2).

Therefore, in FIG. 12, if the point of the lapse of 10 seconds from thepoint when the derivative signal dS/dt fell within the predeterminedrange R1 expressed by formula (2) is within the predetermined period oftime T (200 seconds) after the start of the measurement, the reactioncould be determined as having been completed at an elapsed time of 200seconds. Specifically, for ●, reaction completion could be determined atan elapsed time of 87 seconds. For ▪, reaction completion could bedetermined at an elapsed time of 145 seconds. For X, reaction completioncould be determined at an elapsed time of 176 seconds. By using thedetermination condition of the above example, it was possible toadequately determine whether or not the reaction had been completed.

In measuring the turbidity of a test liquid from the output signal ofthe photosensor 12 to calculate the protein concentration, the outputsignal of the photosensor 12 at the point when the reaction completionwas determined may be analyzed, as described above. Specifically, for ●,the output signal of the photosensor 12 at the elapsed time of 87seconds is used, and for ▪, the output signal of the photosensor 12 atthe elapsed time of 145 seconds is used. For X, the output signal of thephotosensor 12 at the elapsed time of 176 seconds is used. In preparinga calibration curb, the same condition as described above may be usedfor preparation.

FIG. 16 shows the dependency of the output signal of the photosensor 12on the protein concentration. The solid line represents the outputsignal at an elapsed time of 200 seconds, and + represents the outputsignal at an elapsed time of 90 seconds. ▴ represents the output signalat the point when the reaction completion was determined under the abovecondition, i.e., the output signal at the elapsed time of 176 secondsfor 10 mg/dl, the output signal at the elapsed time of 145 seconds for30 mg/dl, and the output signal at the elapsed time of 87 seconds for100 mg/dl. As can be seen from FIG. 16, in the case of the determinationof reaction completion as represented by ▴, the accuracy was retained atthe respective concentrations; however, in the case of the output signalat the elapsed time of 90 seconds as represented by +, the lower theconcentration, the lower the accuracy.

As described above, according to this embodiment, the concentration canbe measured in a necessary and sufficient measurement time while theaccuracy is ensured, so that the measurement time can be shortened.Further, the degradation in accuracy due to insufficient completion ofthe reaction can be avoided.

It is needless to say that the condition for determining the completionof a reaction is not limited to the above-described condition. That is,T, T1, and the predetermined range R1 expressed by formula (2) may beset as appropriate, according to various conditions such as the kind ofthe reagent liquid, the injection speed, the arrangement of the opticalsystem, the accuracy required, the measurement time, the calibrationcurb, etc. Also, in calculating the concentration of a specificcomponent of a test liquid, the computer 7 analyzes the output signal ofthe photosensor 12 at the point when the reaction completion wasdetermined, while referring to the pre-set calibration curb, in order tocalculate the concentration of the test liquid. In preparing acalibration curb, the same condition as described above may be used forpreparation. Also, it may be prepared by grasping the wholecharacteristics of changes over time in output signal responsive tostandard solutions of known concentrations, which corresponds to FIG.10, and using the output signal at the point when reaction completionwas determined.

As described above, according to this embodiment, the degree of reactioncompletion can be determined with the sample cell mounted on the opticalsystem. Further, since the time needed for a measurement is necessaryand sufficient, time can be saved. Accordingly, while the process can besimplified, misoperations are unlikely to occur. These practical effectsare extremely large, thereby making it possible to enhance theefficiency of measurements and tests and to achieve labor-savingsthereof.

This embodiment has described an example in which the turbidity ismeasured by detecting, by means of the photosensor 12, the light that isscattered while the substantially parallel light 4 is propagated throughthe solution. However, when the turbidity is measured as the intensityof transmitted light (the output signal of the photosensor 5), the sameoperations are also possible, and highly accurate measurements can berealized in the same manner.

In this case, however, the higher the protein concentration, the higherthe turbidity, and the smaller the output signal of the photosensor 5.Also, the derivative signal dS1/dt of output signal of the photosensor 5asymptotically approached zero from minus values. In this way, when thereaction completion is determined using the intensity of transmittedlight, T, T1, and the predetermined range R1 expressed by formula (2)may also be set as appropriate, according to various conditions such asthe kind of the reagent liquid, the injection speed, the arrangement ofthe optical system, the accuracy required, the measurement time, thecalibration curb, etc.

Also, the derivative signal of the output signal may be obtained with ananalogue circuit, or, it may be obtained by measuring a plurality oftimes at appropriate intervals of time and performing differentialanalysis. In the above case, the output signal monotonously increased ormonotonously decreased. However, even if it decreases in an oscillatingmanner or increases in an oscillating manner, the methods according tothe present invention are applicable in the same manner.

Embodiment 3

In this embodiment, a different method of determining the completion ofa reaction is described using the apparatus having the structure asillustrated in FIGS. 8 and 9 used in the foregoing Embodiment 2. In thesame manner as in Embodiment 2, the output signal of the photosensor 12as illustrated in FIGS. 10 and 16 was used to calculate and determinethe concentration. However, unlike Embodiment 2, (dS1/dt)/S1 was used inthis embodiment as an index to evaluate whether or not it was in apredetermined range R2.

In Embodiment 2, dS1/dt was used as the evaluation index. In this case,if S1 is relatively small, i.e., if the concentration of a test liquidis in a lower range, dS1/dt itself becomes small. Accordingly, in thecase of a low concentration test liquid, the reaction may be mistakenlydetermined as having been completed even if the degree of reactioncompletion is low. Therefore, in this embodiment, the value obtained bydividing dS1/dt by the output signal S1, i.e., (dS1/dt)/S1, is used asthe evaluation index to determine reaction completion.

First, the maximum period of time within which a measurement isperformed is set in advance. This period refers to the longest waitingperiod of time from the start of a measurement until obtaining theresult, and if a measurement takes more than this maximum period oftime, this measurement is rendered invalid. This pre-set period of timeis referred to as a predetermined period of time T.

According to this method, homogenization is determined when the valueobtained by dividing the amount of change in an output signal S1 of thephotosensor 12 per unit time by the output signal S1, i.e., (dS1/dt)/S1,has continuously been in a predetermined range R2 for a predeterminedperiod of time T2 within the predetermined period of time T.

Specifically, the liquid mixture is determined as having beenhomogenized when (dS1/dt)/S1 [V/S] has continuously been in apredetermined range R2 represented by formula (3) for a predeterminedperiod of time T2 (10 seconds) or longer, within a predetermined periodof time T (200 seconds) after the start of a measurement.−5×10⁻⁴≦(dS1/dt)/S1≦5×10⁻⁴  (3)

In FIG. 13, the value obtained by dividing the derivative signal ofoutput signal of the photosensor 12 by the output signal is plotted inordinate. ●, ▪, and X of FIG. 13 correspond to the values obtained bydividing the derivative signals represented by ●, ▪, and X of FIG. 10,respectively, by the output signals. In FIG. 13, in the same manner asin FIG. 10, during the period of about 2 seconds or more from theelapsed time of 20 seconds at which the injection of the reagentsolution was started, the flux of the injected reagent liquid itselfentered the optical path of the substantially parallel light 4, so thatthe intensity of scattered light was disturbed. As a result, (dS1/dt)/S1changed violently. In FIG. 13, ◯ was omitted, since it looks likesubstantially zero. Since the details of FIG. 13 are not readable, FIG.14 was prepared by enlarging the ordinate around 0 and enlarging theabscissa around 60 to 200 seconds. FIGS. 13 and 14 show that thederivative signal of output signal of the photosensor 12 was in theorder of X>▪>● immediately after the injection of the reagent liquid,and that they asymptotically approached zero.

For the condition for determining the completion of a reaction, a pointof time when the derivative signal dS1/dt/S1 has continuously been inthe predetermined range R2 represented by formula (3) for thepredetermined period of time T1 (10 seconds) or longer within thepredetermined period of time T (200 seconds) after the start of ameasurement was found from FIG. 14 as follows.

(dS1/dt)/S1 became 5×10⁻⁴ [V/S] or less from an elapsed time of 69seconds for ● and from an elapsed time of 148 seconds for ▪. However,for X, even after an elapsed time of 200 seconds, (dS1/dt)/S1 did notfall to 5×10⁻⁴ [V/S] or less. After 200 seconds, ● and ▪ asymptoticallyapproached zero, being in the predetermined range R3 as expressed byformula (3). Therefore, if the point of the lapse of 10 seconds from thepoint when it fell within the predetermined range expressed by formula(3) is within the predetermined period of time T (200 seconds) after thestart of the measurement, the reaction could be determined as havingbeen completed at the point of 200 seconds.

Specifically, for ●, the reaction could be determined as having beencompleted at an elapsed time of 79 seconds. For ▪, the reaction could bedetermined as having been completed at an elapsed time of 158 seconds.For X, however, since the reaction could not be determined as havingbeen completed, this measurement was rendered invalid. Accordingly, asis clear from FIG. 16, when the protein concentration is 10 mg/dl, theaccuracy is low, but such measurement could be rendered invalid. In theforegoing Embodiment 2, even when the accuracy is poor in such a lowconcentration range, the reaction is determined as having beencompleted, and such a measurement is rendered valid, so that thereliability of the measurement may be impaired. In this embodiment,however, by making such measurements invalid, the reliability can besecured.

As described above, according to this embodiment, the concentration canbe measured in a necessary and sufficient measurement time while theaccuracy is ensured, so that the measurement time can be shortened.Further, it is possible to detect the degradation in accuracy due torelatively insufficient completion of the reaction which may occur whenthe test liquid is in a low concentration range, so that the reliabilitycan be improved.

It is needless to say that the condition for determining the completionof a reaction is not limited to the above-described condition. That is,T, T2, and the predetermined range R2 expressed by formula (3) may beset as appropriate, according to various conditions such as the kind ofthe reagent liquid, the injection speed, the arrangement of the opticalsystem, the accuracy required, the measurement time, the calibrationcurb, etc.

Embodiment 4

This embodiment uses the apparatus having the same structure as thatillustrated in FIGS. 8 and 9 used in the foregoing Embodiment 3, butemploys a different method of determining the completion of a reaction.In the same manner as in Embodiment 3, the output signal of thephotosensor 12 as illustrated in FIGS. 10 and 16 is used to calculateand determine the concentration. However, unlike Embodiment 3, thisembodiment also uses an output signal S0 of the photosensor 12 beforethe injection of the reagent liquid. Also, in order to facilitate theunderstanding of S1 values, FIG. 15 showed a graph in which the ordinateof FIG. 12 is expressed logarithmically.

Specifically, instead of the S1 used in Embodiments 2 and 3, S1−S0 isused as an evaluation index. That is, (d(S1−S0)/dt)/(S1−S0) is used asthe index. However, since S0 is evaluated as being independent of time,the equation d(S1−S0)/dt=dS1/dt holds, and whether or not(dS1/dt)/(S1−S0) is in a predetermined range R3 is evaluated.

Therefore, homogenization and/or reaction completion is determined when(dS1/dt)/(S1−S0) has been in the predetermined range R3 for apredetermined period of time T3 within a predetermined period of time T.

Except for the above differences, this method is the same as that ofEmbodiment 3. Accordingly, it is possible to detect the relativedegradation in accuracy in the case of using a low-concentration testliquid, as described in Embodiment 3, without being influenced by theturbidity of the test liquid before the injection of the reagent liquid.

As described above, this embodiment can detect the degradation inaccuracy due to relatively insufficient completion of the reaction whichmay occur in the case of a low-concentration-range test liquid, withoutbeing influenced by the turbidity of the test liquid itself, so that thereliability can be further improved.

Embodiment 5

Referring now to FIG. 17, Embodiment 5 of the present invention isdescribed in detail below. In FIG. 17, the constituent elementsrepresented by reference characters 1 to 12 are the same as theconstituent elements represented by reference characters 1 to 12 in FIG.8, and they function in the same manner. Like the injection port 2, aninjection port 13 is disposed in the side face of the sample cell 1having no optical window and has an internal diameter (diameter) of 0.1cm. A pump 14 injects a reagent liquid into a test liquid in the samplecell 1 from the injection port 13. Also, an arrow 15 indicates theinjection direction in which the reagent liquid is injected from theinjection port 13. In this embodiment, more than one kind of reagentliquid or the like is injected. For example, a buffer solution is firstinjected into the test liquid in the sample cell 1, and then, anantibody reagent liquid or the like is injected.

In this embodiment, following the injection of the buffer solution,which is a first reagent liquid, after the first reagent liquid and thetest liquid are determined as having been homogenized, the opticalproperty of the liquid mixture is measured. Subsequently, the antibodyreagent liquid, which is a second reagent liquid, is injected, and afterthe completion of the reaction, the optical property of the liquidmixture is measured, to determine the concentration. In the following, amethod of determining homogenization and determining reaction completionaccording to the methods described in Embodiments 1 to 4 is describedwith reference to FIG. 18.

First, urine samples (test liquids) having human serum albuminconcentrations of 0.1 mg/dl, 0.3 mg/dl, 1.0 mg/dl, and 3.0 mg/dl wereprepared by adding human serum albumin to urine from which human serumalbumin was not detectable (substantially zero concentration). Also, a0.05 M MOPS buffer solution was prepared as the first reagent solution(R1). Then, an antibody reagent liquid (R2) was prepared as the secondreagent liquid by purifying the antibody component of antihuman albuminrabbit serum.

Thereafter, 0.2 ml of each test liquid was introduced into the samplecell 1, at which point the photosensor 12 started measuring theintensity of scattered light (elapsed time of 0 seconds). At an elapsedtime of 20 seconds, the buffer solution serving as the first reagentsolution (R1) was injected over 2 seconds. Then, using the methoddescribed in any of the above-mentioned embodiments, the first reagentliquid and the test liquid were determined as having been homogenized.At the point when homogenization was determined, the optical property ofthe liquid mixture was measured. Upon the lapse of a predeterminedperiod of time T4 from the point when homogenization was determined, theantibody reagent liquid serving as the second reagent liquid wasinjected over 2 seconds. In other words, during the period of time fromthe point when homogenization was determined until the lapse of thepredetermined period of time T4, the optical property of the liquidmixture was measured to obtain an output signal S0. Then, using themethod described in any of the above-mentioned embodiments, the reactionassociated with the second reagent liquid was determined as having beencompleted, and then the optical property of the liquid mixture wasmeasured to obtain an output signal S1. It was confirmed that thedifference between S0 and S1 thus obtained was proportional to the humanserum albumin concentration of the test liquid.

Likewise, in the case of using more than two kinds of reagent liquids,each reagent liquid is injected, and after the lapse of a predeterminedperiod of time from the point of the determination of homogenization orreaction completion, the next reagent liquid is injected. In each stage,after the point of the determination of homogenization or reactioncompletion and before the injection of the next reagent liquid, theoptical property is measured if necessary, and the concentration iscalculated based on the measured value.

As described above, according to this embodiment, the optical propertyis measured after determining homogenization and/or reaction completionfollowing the injection of each reagent liquid, and thereafter, the nextreagent liquid is injected. Therefore, the respective impacts of thereagent liquids can be distinguished in making measurements, therebyresulting in high reliability. For example, in measuring theconcentration of a specific antigen in a test liquid, the turbidity maybe measured by mixing the test liquid and a buffer solution in advanceand then mixing an antibody reagent liquid. In this case, the turbiditydue to the antibody-antigen combination, which is a specific bindingreaction, can be distinguished from the turbidity of the test liquiditself and the turbidity resulting from the mixing of the test liquidand the buffer solution in making measurements. Accordingly, only thespecific antigen can be specifically detected, and hence, thereliability of measurements can be ensured. It should be noted that thepredetermined period of time T4 is zero or more, and that it may be anyperiod of time during which the optical property can be measured.

INDUSTRIAL APPLICABILITY

As described above, the present invention needs only a period of timenecessary for homogenization and/or completion of a reaction. That is,the invention only requires that the necessary condition be satisfiedwith respect to the measurement time. Therefore, the measurement timecan be shortened, which is highly practically effective, and moreefficient and labor-saving measurements and tests become possible.Further, since measurements with poor accuracy can be rendered invalid,the reliability is high. Also, the concentration can be measured byspecifically detecting only a specific component of a test liquid, whichis greatly practically effective. The invention is applicable to, forexample, urinalysis.

1. A method for determining homogenization and/or reaction completion,comprising the steps of: (1) mixing a test liquid and a reagent liquidto obtain a liquid mixture; (2) measuring an optical property of saidliquid mixture after the mixing continuously or a plurality of timesdiscretely; (3) obtaining a relation between the measured value of theoptical property obtained and the elapsed period of time since the startof the measurement after the mixing; and (4) determining, on the basisof said relation, whether said test liquid and said reagent liquid havebeen substantially homogeneously mixed with each other and/or a reactionbetween said test liquid and said reagent liquid has been substantiallycompleted, wherein said step (3) is a step of obtaining (dS1/dt)/S1(wherein S1 is the measured value of the optical property obtained and Tis the elapsed period of time since the start of the measurement afterthe mixing), and said step (4) is a step of determining that said testliquid and said reagent liquid have been substantially homogeneouslymixed with each other and/or the reaction between said test liquid andsaid reagent liquid has been substantially completed, when the(dS1/dt)/S1 has continuously been in a predetermined range R2 for apredetermined period of time T2 or longer.
 2. The method for determininghomogenization and/or reaction completion in accordance with claim 1,wherein a measurement is rendered invalid when homogenization and/orreaction completion has not been determined within a predeterminedperiod of time T from the start of the measurement.
 3. A method fordetermining homogenization and/or reaction completion, comprising thesteps of: (1) mixing a test liquid and a reagent liquid to obtain aliquid mixture; (2) measuring an optical property of said test liquidand said liquid mixture continuously, or, measuring an optical propertyof said test liquid at least once and measuring an optical property ofsaid liquid mixture after the mixing a plurality of times discretely;(3) obtaining a relation between the measured value of the opticalproperty obtained and the elapsed period of time since the start of themeasurement after the mixing; and (4) determining, on the basis of saidrelation, whether said test liquid and said reagent liquid have beensubstantially homogeneously mixed with each other and/or the reactionbetween said test liquid and said reagent liquid has been substantiallycompleted, wherein said step (3) is a step of obtaining (dS1/dt)/(S1-S0)(wherein S0 is the measured value of the optical property of said testliquid, S1 is the measured value of the optical property of said liquidmixture, and T is the elapsed period of time since the start of themeasurement after the mixing), and said step (4) is a step ofdetermining that said test liquid and said reagent liquid have beensubstantially homogeneously mixed with each other and/or the reactionbetween said test liquid and said reagent liquid has been substantiallycompleted, when the (dS1/dt)/(S1-S0) has continuously been in apredetermined range R3 for a predetermined period of time T3 or longer.4. The method for determining homogenization and/or reaction completionin accordance with claim 3, wherein a measurement is rendered invalidwhen homogenization and/or reaction completion has not been determinedwithin a predetermined period of time T from the start of themeasurement.
 5. A method for measuring solution concentration,comprising the steps of: (1) mixing a test liquid and a reagent liquidto obtain a liquid mixture; (2) measuring an optical property of theliquid mixture after the mixing continuously or a plurality of timesdiscretely; (3) obtaining a relation between the measured value of theoptical property obtained and the elapsed period of time since the startof the measurement after the mixing; (4) determining, on the basis ofsaid relation, whether said test liquid and said reagent liquid havebeen substantially homogeneously mixed with each other and/or a reactionbetween said test liquid and said reagent liquid has been substantiallycompleted; and (5) determining the concentration of a specific componentof said test liquid based on said measured value, wherein said step (3)is a step of obtaining (dS1/dt)/S1 (wherein S1 is the measured value ofthe optical property obtained and T is the elapsed period of time sincethe start of the measurement after the mixing), and said step (4) is astep of determining that said test liquid and said reagent liquid havebeen substantially homogeneously mixed with each other and/or thereaction between said test liquid and said reagent liquid has beensubstantially completed, when the (dS1/dt)/S1 has continuously been in apredetermined range R2 for a predetermined period of time T2 or longer.6. The method for measuring solution concentration in accordance withclaim 5, further comprising the step of mixing another reagent liquidwith said test liquid, after determining that the said test liquid andsaid reagent liquid have been homogeneously mixed and/or the reactiontherebetween has been substantially completed.
 7. The method formeasuring solution concentration in accordance with claim 6, whereinanother reagent liquid is mixed with said test liquid upon the lapse ofa predetermined period of time T4 after determining that the said testliquid and said reagent liquid have been homogeneously mixed and/or thereaction therebetween has been substantially completed, and the opticalproperty of said liquid mixture is measured prior to the lapse of thepredetermined period of time T4.
 8. The method for measuring solutionconcentration in accordance with claim 5, wherein a measurement isrendered invalid when homogenization and/or reaction completion has notbeen determined within a predetermined period of time T from the startof the measurement.
 9. A method for measuring solution concentration,comprising the steps of: (1) mixing a test liquid and a reagent liquidto obtain a liquid mixture; (2) measuring an optical property of saidtest liquid and said liquid mixture continuously, or, measuring anoptical property of said test liquid at least once and measuring anoptical property of said liquid mixture after the mixing a plurality oftimes discretely; (3) obtaining a relation between the measured value ofthe optical property obtained and the elapsed period of time since thestart of the measurement after the mixing; (4) determining, on the basisof said relation, whether said test liquid and said reagent liquid havebeen substantially homogeneously mixed with each other and/or a reactionbetween said test liquid and said reagent liquid has been substantiallycompleted; and (5) determining the concentration of a specific componentof said test liquid based on said measured value, wherein said step (3)is a step of obtaining (dS1/dt)/(S1-S0) (wherein S0 is the measuredvalue of the optical property of said test liquid, S1 is the measuredvalue of the optical property of said liquid mixture, and T is theelapsed period of time since the start of the measurement after themixing), and said step (4) is a step of determining that said testliquid and said reagent liquid have been substantially homogeneouslymixed with each other and/or the reaction between said test liquid andsaid reagent liquid has been substantially completed, when the(dS1/dt)/(S1-S0) has continuously been in a predetermined range R3 for apredetermined period of time T3 or longer.
 10. The method for measuringsolution concentration in accordance with claim 9, further comprisingthe step of mixing another reagent liquid with said test liquid, afterdetermining that the said test liquid and said reagent liquid have beenhomogeneously mixed and/or the reaction therebetween has beensubstantially completed.
 11. The method for measuring solutionconcentration in accordance with claim 10, wherein another reagentliquid is mixed with said test liquid upon the lapse of a predeterminedperiod of time T4 after determining that the said test liquid and saidreagent liquid have been homogeneously mixed and/or the reactiontherebetween has been substantially completed, and the optical propertyof said liquid mixture is measured prior to the lapse of thepredetermined period of time T4.
 12. The method for measuring solutionconcentration in accordance with claim 9, wherein a measurement isrendered invalid when homogenization and/or reaction completion has notbeen determined within a predetermined period of time T from the startof the measurement.